Toxic proteins are defined by their negative effect on cell viability and therefore cloning and expressing of such toxic genes have proved problematic. One approach to this problem has been to generate cell-based expression systems that are inducible. The theory is that gene expression is inhibited in host cells until the cell density has reached the desired concentration at which time the inhibition of protein expression is reversed and protein is expressed up to a threshold amount before viability is compromised.
An example of an inducible cell system is BL21(DE3), This is an E. coli host strain in which the bacteriophage DE3, which is a lambda derivative, has been incorporated into the host cell chromosome, the DE3 prophage encodes the gene for T7 RNA polymerase, which is regulated by the lacUV5 promoter and controlled by LacI repression (U.S. Pat. No. 4,952,496). In such DE3 lysogens, the target gene is typically expressed from a multi-copy plasmid by transcription from a T7 promoter. Thus, induction of target gene expression can be activated by IPTG or allolactose. Unfortunately this system permits a significant amount of basal expression of toxic protein under LacI repression and therefore significantly compromises clone stability and yield of protein.
Various attempts have been mode to reduce basal expression, (See for example, Dubendorff and Studier, J. Mol. Biol. 219:45-59 (1991); Moffatt and Studier, Cell 49:221-227 (1987); Studier J. Mol. Biol. 219:37-44 (1991)). These modifications include expressing the target gene from a T7-lac promoter. The T7-lac promoter consists of a lac operator sequence inserted just downstream of the T7 φ10 promoter (beginning with vector pET-10). This enables the Lac repressor to bind at the transcriptional start site and function to reduce transcription in the absence of induction. In such T7 lac vectors, the vector also encodes lacI to provide a greater supply of Lac repressor.
Another modification is the introduction of a multi-copy plasmid that contains a gene expressing an inhibitor of the T7 RNA polymerase, in particular, wild type (WT) T7 lysozyme (pLysS, pLysE, pLysL, and pLysH). In pLysS and pLysL, lysozyme expression is constitutively expressed by readthrough transcription from the CAT promoter. In pLysE and pLysH, lysozyme is expressed at a higher level from the tet promoter.
U.S. Pat. No 6,569,669 describes a host cell with an increased concentration of lac repressor (greater than about 10 molecules per cell) to control the expression of a foreign RNA polymerase.
The above attempted solutions have some disadvantages. These include the following:
(a) Even when it is desirable to induce target gene expression, the constitutive production of T7 lysozyme from multi-copy plasmids results in a lag time after induction before a sufficient level of T7 RNA polymerase is produced to effect gene expression. This is especially significant when using pLysE for expression of extremely toxic genes. This expression lag is problematic because there is a limited window of maximal protein production during the exponential growth phase of a typical fermentation.
(b) Maintenance of the multi-copy plasmids requires the addition of chloramphenicol (CAM) into the growth media of the host cells that results in significant expense for large-scale fermentations.
(c) The second multi-copy plasmid may interfere with the characterization of the multi-copy plasmid expressing the target protein from the host cells.
(d) The expression of large amounts of secondary proteins from the multi-copy plasmids (such as lysozyme and antibiotic resistance proteins, e.g. CAT) puts a burden on the protein translation apparatus that might otherwise make larger amounts of the target protein.
(e) The expression of active T7 lysozyme within a protein expression strain may also interfere with post expression cell processing and with certain experimental procedures. For example, pLysS and pLysE strains often lyse spontaneously upon freezing and thawing.
(f) The co-expression of active T7 lysozyme during target membrane protein over-expression may result in cell lysis and a reduction in culture yield.